Cost-Effective Real-Time Metabolic Profiling of Cancer Cell Lines for Plate-Based Assays

Round 1

Reviewer 1 Report

Dear Authors,

Very interesting topic but you need to add some points:

1. Line 39-49 - I don't see citation information.
2. Line 64 - what kind of plates did you use? Flat or U-bottom?
3. Materials and methods have to be improved. Unfortunately, the information contained are very general: how dissolved HPTS was stored? "Salts for preparing media were obtained from SA", What was added to DMEM and RPMI? Did you culture all cell types only in RPMI? How did you prepare cells? Were they frozen? Line 80 - I understand that passage number for AsPC1 passage number was 3, etc? it should be clear. 
4. Line 101: what was used for titration?
5. Did you try to check your method also for other types of cells? Or only cancer?

Please, describe very carefully cell preparation procedure - exactly number of cells per well, which much medium did you use, how media was complemented etc. Such information makes it easier for others to repeat the tests described.

Author Response

We thank Reviewer 1 for the critique on how to improve our manuscript. Below, we provide a point-by-point response. 

1. Line 39-49 - I don't see citation information. We have now added additional references throughout this paragraph.
2. Line 64 - what kind of plates did you use? Flat or U-bottom? Now added (flat)
3. Materials and methods have to be improved. Unfortunately, the information contained are very general: how dissolved HPTS was stored? "Salts for preparing media were obtained from SA", What was added to DMEM and RPMI? Did you culture all cell types only in RPMI? How did you prepare cells? Were they frozen? Line 80 - I understand that passage number for AsPC1 passage number was 3, etc? it should be clear. Thank you for raising these inadequacies in the text. We have now expanded the methods accordingly.
4. Line 101: what was used for titration? This information has now been added.
5. Did you try to check your method also for other types of cells? Or only cancer? This is a very good point.  In principle, the assay can be used in any type of cell that has a sufficient metabolic rate to affect pH and/or O2. This included primary, non-adherent cells. As proof-of-principle, we have performed experiments on primary cardiac myocytes isolated from a mouse heart. The data are presented in Fig 9.

Please, describe very carefully cell preparation procedure - exactly number of cells per well, which much medium did you use, how media was complemented etc. Such information makes it easier for others to repeat the tests described. We appreciate this comment and have expanded the details throughout the Methods and Results. We also added a section after the Discussion, on the protocol, providing a step-by-step guide. 

Reviewer 2 Report

This manuscript by Blaszczack et al. presents a novel metabolic assay that enables measurement of proton and oxygen fluxes of cancer cells. The assay developed in this study is cost-effective and easily adoptable because it features standard culture plates, fluorescence microplate reader, and low-cost commercial dyes. The assay development procedure is very thorough and comprehensive. The authors empirically determined the measurement settings that maximize sensitivity to pH and minimize crosstalk between pH and oxygen channels. They also accounted for oxygen equilibration rate and the buffering capacity of the medium to establish a calibration protocol that converts fluorescence intensities to total proton and oxygen concentrations. The measured proton and oxygen concentrations and fluxes were shown to depend on cell type, cell density, media, and the presence of metabolic inhibitors. Overall, this manuscript introduces a cost-effective, powerful assay for metabolic phenotyping of cancer cells and drug screening. I recommend it for publication.

Some minor points: 

The loading rate of the celltracker dyes might depend on the physiological state of the cell (pH and oxygen concentration), in which case CTO might not be a good proxy for the cell number. Is there any evidence that shows that CTO intensity per cell is relatively insensitive to pH or oxygen concentration?

P3.141: Figure 1D -> Figure 1E

P3.132: Ch3 and Ch4 should be switched (see Figure 1E, P3.142, and P3.145).

Author Response

We thank Reviewer 2 for the comments.

1. CTO pH/O2 sensitivity. Thank you for raising this point.  We have now tested CTO and determined this to be pH and O2 insensitive. The data are shown in Fig 5E and F. This is consistent with the chemistry of CellTracker dyes, designed to be robust fluorescent probes.

2. Thank you for pointing out the typos in the text. We have now corrected these.